Acetylation of prb by p300 assay method for compounds which modulate this process

ABSTRACT

The present invention is based upon the finding that the protein p300 has acetylation activity which is directed to the retinoblastoma tumour suppressor protein pRb by the presence in the cell of the adenovirus E1A protein. This represents a target for modulators of the cell cycle, to which end the invention provides an assay for a modulator of acetylation of pRb by p300, which comprises: a) bringing into contact a p300 protein a pRb protein and a putative modulator compound under conditions where the p300 protein, in the absence of said modulator is capable of acetylating the pRb protein; b) providing conditions for acetylation of said pRb protein; and c) measuring the degree of inhibition of acetylation caused by said modulator compound.

[0001] The present invention relates to the finding of a novelinteraction between the proteins p300 and pRb, assays based upon thisinteraction and novel compounds obtainable by such assay methods.

BACKGROUND TO THE INVENTION.

[0002] The product of the retinoblastoma tumour suppressor gene, pRb,mediates control of the G1 to S phase transition by interacting withgrowth-regulating transcription factors, such as the E2F family [1, 2].The Rb gene is frequently mutated in tumour cells, and can beinactivated through the physical association with viral oncoproteinssuch as adenovirus E1A [3]. Post-translational phosphorylation controlof pRb by G1 cyclin-dependent kinases plays an important role inregulating pRb activity [4]. The p300/CBP transcriptional co-activatorproteins are endowed with histone acetyltransferase (HAT), which isinvolved in regulating chromatin [6].

DISCLOSURE OF THE INVENTION

[0003] We have identified acetylation as a new level of control in theregulation of pRb. Adenovirus E1A, which sequesters p300/CBP proteinsthrough an N-terminal transformation-sensitive domain3, stimulates theacetylation of pRb by the E1A-dependent recruitment of p300 and pRb intoa ternary protein complex. Our data indicate that the acetylation of pRbis specific to the p300/CBP histone acetyl transferases and not afeature of other HATs such as pCAF.

[0004] We also observed that the MDM2 oncoprotein can overcome thecontrol of E2F by pRb [7, 8], and our results show that MDM2preferentially binds to the acetylated form of pRb. Furthermore,acetylation and phosphorylation appear to be distinct levels of controlthat act on pRb activity. The acetylation of pRb by p300, and theinfluence on the interaction between MDM2 and pRb, defines a new levelof cell cycle control. Moreover, these observations establish a novelrelationship between p300, pRb and acetylation in which oncoprotein E1Aacts to recruit and target a cellular HAT activity to pRb to effect theloss of pRb-dependent growth control.

[0005] Thus, the tumour suppressor pRb is acetylated by the p300protein, and this acetylation has downstream consequences on theactivity of pRb, both in its ability to be phosphorylated by proteinkinases, and its ability to interact with the protein MDM2. Inconsequence, the acetylation of pRb appears to be an event which leadsto cell cycle progression so that inhibition of this event provides atarget for inhibition of cell proliferation.

[0006] Thus in a first aspect the present invention provides an assayfor a modulator of acetylation of pRb by p300, which comprises:

[0007] a) bringing into contact a p300 protein, a pRb protein and aputative modulator compound under conditions where the p300 protein, inthe absence of said modulator is capable of acetylating the pRb protein;and

[0008] b) providing conditions for acetylation of said pRb protein; and

[0009] c) measuring the degree of inhibition of acetylation caused bysaid modulator compound.

[0010] In a further aspect, the invention provides compounds obtainableby such an assay, for example peptide compounds based on the portions ofp300 or pRb which interact with each other in order to provideacetylated pRb.

[0011] The assay of the invention may be performed in vitro usingisolated, purified or partially purified p300 and pRb proteins, or incell free or cellular systems.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIGS. 1a-e shows that the retinoblastoma protein is modified byacetylation.

[0013]FIGS. 2a-f shows that the C-terminal region of pRb is a major siteof acetylation.

[0014]FIGS. 3a-c show adenovirus E1A augments the p300-dependentacetylation of the pRb protein.

[0015]FIGS. 4a-g shows that domains in E1A for p300-dependentacetylation of pRb and acetylated pRb binds to MDM2.

[0016]FIGS. 5a-b shows functional effects of pRb acetylation.

[0017]FIG. 6 is an overview of acetylation control of pRb activity.

DETAILED DESCRIPTION OF THE INVENTION

[0018] P300.

[0019] “p300” is a co-activator protein which has histoneacetyltransferase activity, and has been widely studied in the art. p300is described for example by Eckner et al, 1994 (Genes Dev., 8(8);869-84). For the purposes of the present invention, reference to p300refers to human allelic and synthetic variants of p300, and to othermammalian variants and allelic and synthetic variants thereof, as wellas fragments of said human and mammalian forms of p300, and its relatedfamily member CBP (reviewed in R H. Goodman and S Smolik Genes Dev.(2000) 14: 1553-1577.) Reference herein to p300 is intended to includep300/CBP unless the context is clearly otherwise.

[0020] Human p300 has been cloned and sources of the gene can be readilyidentified by those of skill in the art. Its sequence is available asGenbank accession numbers A54277. Other mammalian p300s are alsoavailable, such as murine p300 (accession number AAB31182). P300 genesequences may also be obtained by routine cloning techniques, forexample by using all or part of the human p300 gene sequence as a probeto recover and to determine the sequence of the p300 gene in otherspecies. A wide variety of techniques are available for this, forexample PCR amplification and cloning of the gene using a suitablesource of mRNA (e.g. from an embryo or a liver cell), obtaining a cDNAlibrary from a mammalian, vertebrate, invertebrate or fungal source,e.g. a cDNA library from one of the above-mentioned sources, probingsaid library with a polynucleotide of the invention under stringentconditions, and recovering a cDNA encoding all or part of the p300protein of that mammal.

[0021] Suitable stringent conditions include hybridization on a solidsupport (filter) overnight incubation at 42 C in a solution containing50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulphate and 20μg/ml salmon sperm DNA, followed by washing in 0.03M sodium chloride and0.03M sodium citrate (i.e. 0.2×SSC) at from about 50 C to about 60 C).Where a partial cDNA is obtained, the full length coding sequence may bedetermined by primer extension techniques.

[0022] A further approach is to use the above-identified sequences asquery sequences to search databases for homologous gene sequences orpartial gene sequences (particularly ESTs). Matches identified may beexamined and where an actual or putative p300 sequence is found, thegene recovered by physical cloning using, for example PCR and RACE-PCRbased on the sequence of the match.

[0023] Although wild-type p300 is preferred, and variants of p300 whichstill retain the ability to acetylate the pRb may also be used. Suchvariants will generally be based on wild-type mammalian p300s and have adegree of amino acid identity which is desirably at least 70%,preferably at least 80%, 90%, 95% or even 98% identity to a wild typemammalian p300, preferably to human p300.

[0024] It is not necessary to use the entire p300 proteins (or variantsthereof) for assays of the invention. Fragments of the p300 may-be usedprovided such fragments retain the ability to acetylate the targetdomain of the pRb. Examples of two such fragments, p3001195-1673 andp3001135-2414 are provided in the accompanying examples. Thisdemonstrates that a fragment of at least about 450 amino acids or more,such as at least about 1000 amino acids can be made with this ability.Those of skill in the art will be able to construct similar fragmentsusing routine skill and knowledge, e.g. by PCR subcloning, in a manneranalogous to that illustrated below for the production of pRb fragments.

[0025] The ability of suitable variants or fragments to acetylate pRb(or a fragment thereof) may be tested using routine procedures such asthose described in the accompanying examples.

[0026] Reference herein to a p300 protein includes the above-mentionedvariants and fragments which are functionally able to acetylate pRbunless the context is explicitly to the contrary.

[0027] pRb Protein.

[0028] The pRb protein is a pocket protein involved in the regulation ofthe cell cycle and has been widely studied in the art. pRb is describedfor example by Lee et al, 1987, Science, 235(4794); 1394-9. For thepurposes of the present invention, reference to pRb refers to humanallelic and synthetic variants of pRb, and to other mammalian variantsand allelic and synthetic variants thereof, as well as fragments of saidhuman and mammalian forms of pRb.

[0029] Human pRb has been cloned and sources of the gene can be readilyidentified by those of skill in the art. Its sequence is available asGenbank accession number P06400. Other pRbs are also available, such asmurine pRb (Genbank accession number P13405), rat pRB (Genbank P33568),chicken (CAA51019) and salamander (CAA70428). pRb gene sequences mayalso be obtained by routine cloning techniques, for example by using allor part of the human pRb gene sequence as a probe to recover and todetermine the sequence of the pRb gene in other species. A wide varietyof techniques are available for this, for example PCR amplification andcloning of the gene using a suitable source of mRNA (e.g. from an embryoor a liver cell), obtaining a cDNA library from a mammalian, vertebrate,invertebrate or fungal source, e.g a cDNA library from one of theabove-mentioned sources, probing said library with a polynucleotide ofthe invention under stringent conditions, and recovering a cDNA encodingall or part of the pRb protein of that mammal.

[0030] Suitable stringent conditions include those described above inrelation to p300. Where a partial cDNA is obtained, the full lengthcoding sequence may be determined by primer extension techniques.

[0031] A further approach is to use the above-identified sequences asquery sequences to search databases for homologous gene sequences orpartial gene sequences (particularly ESTs), as described above inrelation to p300.

[0032] Although wild-type pRb is preferred, and variants of pRb whichstill retain the ability to be acetylated by p300 may also be used. Suchvariants will generally be based on wild-type mammalian pRbs and have adegree of amino acid identity which is desirably at least 70%,preferably at least 80%, 90%, 95% or even 98% identity to a wild typemammalian pRb, preferably to human pRb.

[0033] It is not necessary to use the entire pRb proteins (or variantsthereof) for assays of the invention. Fragments of the pRb may be usedprovided such fragments retain the ability to be acetylated by p300. Ourdata indicate that the C-terminal region of pRb is the target foracetylation by the p300 protein. Thus as exemplified in the accompanyingexamples, fragments which contain this region may be used in assays ofthe invention. Examples of such fragments include those in theaccompanying examples spanning the residues lysine 873 and 874 of thehuman pRb sequence. This demonstrates that fragments comprising thesetwo residues which are as little as about 40 amino acids or more, suchas 50, 60 or 80 amino acids or more can be made and used in an assay ofthe invention. There is also evidence that similar sized fragmentscomprising the region 794-829 may be acetylated. In any event, suchfragments will comprise at least one target lysine residue foracetylation.

[0034] Those of skill in the art will be able to construct similarfragments using routine skill and knowledge, e.g. by PCR subcloning, ina manner analogous to that illustrated below for the production of pRbfragments. The ability of suitable variants or fragments to beacetylated by p300 (or a fragment thereof) may be tested using routineprocedures such as those described in the accompanying examples.Reference herein to a pRb protein includes the above-mentioned variantsand fragments which are functionally able to be acetylated by pRb unlessthe context is explicitly to the contrary.

[0035] The assays of the invention preferably use the same mammaliansource pRb as the p300.

[0036] p53

[0037] p53 refers to the tumour suppressor gene or its encoded aminoacid sequence of as reported, for example, by Matlashewski et al (EMBOJ. 13; 3257-62, 1984) or Lamb and Crawford (Mol. Cell. Biol. 5; 1379-85,1986). These sequences are available on Genbank. Wild-type human p53protein includes a proline/arginine polymorphism at amino acid 72,reflecting a corresponding polymorphism in the gene. Reference to p53further includes mutated forms of p53 as found in many tumour cells.Such mutations include point mutations, for example from 1 to 10, e.gfrom 1 to 5 point mutations (which point mutations result in a change tothe amino acid sequence) to the wild-type sequences. It further includesfragments of wild-type and mutated p53 which retain the ability to be asubstrate for acetylation by p300. Such fragments are preferably atleast 20, more preferably at least 30 and most preferably at least 50amino acids in size.

[0038] MDM2

[0039] The sequence of the human MDM2 oncoprotein may be found byreference to Genbank accession number Q00987. Other mammalian MDM2proteins are known, for example mouse (Genbank P23804), horse (P56273),dog (P56950) and hamster (Q60624), as well as non-mammalian homologuessuch as chicken (AAF04192), xenopus (P56273) and zebrafish (O42354).Where assays of the invention involve the use of an MDM2 oncoprotein,this may be a wild-type MDM2 or a variant which is capable of binding toacetylated pRB. Such variants will generally be based on wild-typemammalian MDM2s and have a degree of amino acid identity which isdesirably at least 70%, preferably at least 80%, 90%, 95% or even 98%identity to a wild type mammalian MDM2, preferably to human MDM2. Theseinclude variants obtainable from other species, in a manner analogous tothat described above for p300 and pRb.

[0040] Amino Acid Identity.

[0041] The percentage homology (also referred to as identity) of DNA andamino acid sequences can be calculated using commercially availablealgorithms. The following programs (provided by the National Center forBiotechnology Information) may be used to determine homologies: BLAST,gapped BLAST and PSI-BLAST, which may be used with default parameters.The algorithm GAP (Genetics Computer Group, Madison, Wis.) uses theNeedleman and Wunsch algorithm to align two complete sequences thatmaximizes the number of matches and minimizes the number of gaps.Generally, the default parameters are used, with a gap creationpenalty=12 and gap extension penalty=4. Use of either of the terms“homology”-and “homologous” herein does not imply any necessaryevolutionary relationship between compared sequences, in keeping forexample with standard use of terms such as “homologous recombination”which merely requires that two nucleotide sequences are sufficientlysimilar to recombine under the appropriate conditions.

[0042] Where default parameters or other features of these programs aresubject to revision, it is to be understood that reference to theprograms and their parameters are as of the priority date of the instantapplication.

[0043] Assay formats.

[0044] Assays according to the present invention may be provided in anysuitable format. Desirably, the assays will be adapted to provide for ahigh throughput format, such that a large number of putative modulatorcompounds may be screened simultaneously and/or sequentially, usingautomated robotic technology which is available in the art formethodology of this nature. Assays of the invention may be used toscreen for inhibitors of acetylation, in which case the measured degreeof acetylation will be reduced by the presence of the modulator whencompared to a control in which the modulator is absent.

[0045] In one format, labelled acetyl CoA is provided together with p300and pRb proteins in appropriate concentrations in a buffer whichprovides for acetylation in the absence of an inhibitory modulator. Theproteins are incubated in the presence of a putative modulator and theamount of acetylation is determined, e.g. by recovering the pRb proteinand observing or measuring the amount of labelled acetate incorporatedinto the pRb.

[0046] Alternatively, the acetylation of pRb may be determined usingantibodies which bind to acetylated lysine residues on the pRb. Suchantibodies are commercially available, including those used in theaccompanying examples.

[0047] The amount of acetylation in the presence of a modulator may becompared to the amount obtained in the absence of a modulator. Such acontrol may be performed in parallel with the assay, or be astandardised control value to which the amount is compared. In a highthroughput format, the skilled person may opt to dispense with anegative (i.e. no modulator present) control and instead select apre-determined percentage of the compounds screened, wherein thesecompounds are at the top end of the distribution of activity of all thecompounds in a set of compounds being screened.

[0048] The assay of the invention may also be conducted in cells inculture or in cell lysates. In such circumstances, the cells may expressp300 or pRb naturally, or contain a recombinant DNA construct from whichthe p300 or pRb is expressed.

[0049] Desirably, at least the pRb protein is in the form of a fusionprotein comprising the pRb and a detectable tag, such as GST or a histag (many others are known as such in the art), so that the pRb may beisolated via the tag—e.g. by immuno-precipitation or usingglutathione-Sepharose (TM) beads, thus allowing accurate quantitation ofpRb acetylation.

[0050] There are a number of additional components which may be used inthe assays of the invention. For example, we have found that theadenovirus protein E1A, which binds both p300 and pRb, provides forenhanced acetylation of pRb by p300, at least up to a concentration ofE1A which favours the formation of a ternary E1A-p300-pRb complex overbinary E1A-p300 and E1A-pRb complexes. Thus E1A may be provided toassays of the invention in order to enhance the acetylation of pRb. TheE7 protein of HPV is also known to bind to both p300 and pRb, and mayalso be used to provide a similar effect. The HPV E7 protein may be fromany HPV species, particularly those associated with cervical cancer,such as HPV 16. Similarly, other viral oncoproteins which bind at leastone of p300 or pRb, preferably both, may also be used.

[0051] Moreover, the up-regulation in acetylation of pRb by theformation of a ternary complex may be a normal cellular process.Accordingly, a cellular protein may be used in place of these viraloncoproteins to achieve the same effect. For example, we have found thatthe cellular protein “JMY” described in SEQ ID NO:2 of WO99/20752, thedisclosure of which is incorporated herein by reference, binds p300 in aregion which is also responsible for E1A binding. JMY may be used inplace of E1A to provide a binding complex of p300, pRb and JMY.

[0052] We have also found that pRb when acetylated binds to the MDM2oncoprotein more strongly than when not acetylated. Thus the assay ofthe invention may incorporate MDM2 and the output of the assay may bemeasured as the amount of MDM2-pRb binding observed followingacetylation of pRb by p300 in the presence or absence of a putativemodulator. The MDM2-pRb binding may be determined in any suitable way,for example by immuno-precpitation. precipitation.

[0053] The interaction between acetylated pRb and MDM2 gives rise to afurther aspect of the invention, since this interaction provides afurther target for inhibition of cell cycle progression. Thus there isalso provided by the invention an assay for a modulator of cell cycleprogression which comprises:

[0054] a) bringing into contact an acetylated pRb protein and an MDM2protein under conditions where, in the absence of modulator, the twoproteins bind to each other;

[0055] b) providing a putative modulator; and

[0056] c) determining the degree of modulation of binding of the twoproteins caused by said modulator.

[0057] By “acetylated pRb” it is meant a pRb protein as defined hereinwherein at least one lysine residue is acetylated.

[0058] We have also found that acetylation of pRb influences the abilityof the cyclin dependent kinase cyclin E/cdk2 to phosphorylate pRb. Thusthe amount of acetylation of pRb may be determined indirectly by asubsequent phosphorylation assay, for example by bringing the pRb intocontact with cyclin E/cdk2 in the presence of gamma 32P labelled ATP,followed by separation of the pRb and the determination of the amount oflabelled phosphate incorporated therein.

[0059] In another aspect, we have found that a consequence of E1Abinding p300 is that the ability of p300 to acetylate p53 and E2F-1 iscompromised. Since p53 is activated by acetylation, the binding of p300to E1A and the consequent redirection of p300 HAT activity to pRb whichresults points to a mechanism by which oncoproteins can antagonise thefunction of p53.

[0060] This finding may be used to provide an assay in which p53acetylation is measured as an end point in an assay for modulators ofthe interaction between p300 and onconproteins which bind p300,particularly E1A.

[0061] Modulator compounds.

[0062] The amount of putative modulator compound which may be added toan assay of the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.01 to100 nM concentrations of putative modulator compound may be used, forexample from 0.1 to 10 nM. Modulator compounds may be those which eitheragonise or antagonise the interaction. Antagonists (inhibitors) of theinteraction are particularly desirable.

[0063] Modulator compounds which may be used may be natural or syntheticchemical compounds used in drug screening programmes. Extracts of plantswhich contain several characterised or uncharacterised components mayalso be used. Many libraries of natural and synthetic compounds arecommercially available for drug screening programs, and these may bebought and used in the assays of the invention.

[0064] Antibodies directed to the site of interaction in either proteinform a further class of putative inhibitor compounds. Candidateinhibitor antibodies may be characterised and their binding regionsdetermined to provide single chain antibodies and fragments thereofwhich are responsible for disrupting the interaction between p300 andthe pRb.

[0065] Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of p300 and pRb and using rational drug designto provide potential inhibitor compounds with particular molecularshape, size and charge characteristics. Assays of the invention andmodulator compounds of the invention have a variety of uses. Forexample, the task of dissecting the complex pathways of cellularproliferation will be facilitated by the provision of means to promoteor inhibit a specific interaction, allowing the effects of otherproteins in the pathway to be studied in better detail.

[0066] Candidate modulator compounds obtained according to the method ofthe invention may be prepared as a pharmaceutical preparation. Suchpreparations will comprise the compound together with suitable carriers,diluents and excipients. Such formulations form a further aspect of thepresent invention.

[0067] Formulations may be prepared suitable for any desired route ofadministration, including oral, buccal, topical, intramuscular,intravenous, subcutaneous and the like.

[0068] Formulations for topical administration to the skin may includeingredients which enhance the permeability of the skin to the peptides.Such formulations may be in the form of ointments, creams, transdermalpatches and the like.

[0069] Formulations for administration by injection (i.m., i.v.,subcutaneous and the like) will include sterile carriers such asphysiological saline, optionally together with agents which preserve orstabilise the peptide. Albumin may be a suitable agent.

[0070] Formulations of inhibitor compounds in particular may be used inmethods of treatment, such as controlling cell proliferation in diseasestates such as cancer, pre-cancerous cell growth, psoriasis, etc.Compounds obtainable by such an assay form a further aspect of theinvention.

[0071] All publications, patent applications and sequence accessiondisclosures cited in this specification are herein incorporated byreference as if each individual publication, patent application orsequence were specifically and individually indicated to be incorporatedby reference.

[0072] The following examples illustrate the invention.

[0073] A. Acetylation of pRb In Vitro.

[0074] Some co-activators, such as p300/CBP, are endowed with HATactivity5, 6. To evaluate whether pRb could be acetylated by the p300HAT, we assessed if a GST-pRb fusion protein was acetylated in vitro. Wefound that GST-pRb could be acetylated by p300 in a fashion that isinfluenced by the integrity of the pocket domain, as the acetylation oftwo pocket mutants (pRbΔ21 and Δ22) isolated from human tumour cells 9was very much reduced (FIGS. 1a and b). A similar analysis usingenzymatically active P/CAF10 failed to provide evidence for pRbacetylation mediated by the P/CAF HAT activity, suggesting thatacetylation of pRb was HAT-specific.

[0075] B. Acetylation of pRb in SAOS2 cells.

[0076] Whilst the above results indicate that pRb can be acetylated, theanalysis was based on the effects of the HAT activity observed in vitroand, therefore, it was important to establish that pRb could beacetylated in vivo under physiological conditions. In order to assesspRb acetylation, we used an antibody directed against acetylated lysineswhich specifically recognised pRb that had been acetylated by the p300HAT, but failed to bind to unacetylated pRb (FIG. 1c).

[0077] We used this antibody to assess pRb acetylation in cells. To thisend, pRb was immunoprecipitated from SAOS2 (Rb−/−) cells transfectedwith a pRb expression vector, and the immunoprecipitate immunoblottedwith the anti-acetylated lysine antiserum, upon which acetylated pRb wasapparent (FIG. 1d). Taking a similar approach, this conclusion wassubstantiated in non-transfected 293 cells where endogenous acetylatedpRb was clearly detected (FIGS. 1e and 4 d). Overall, these resultsestablish that pRb is physiologically acetylated.

[0078] C. Acetylation of pRb is in the C-Terminal Region.

[0079] Whilst the acetylation of pRb required the integrity of thepocket region (FIG. 1a), it was nevertheless possible that acetylationin the wild-type protein occurred outside the pocket region, and thus wedetermined more precisely the region in pRb that could be directlytargeted by acetylation. To assess the general region in pRb that isacetylated by p300, we examined the in vitro acetylation status of thetwo halves of the pocket, together with the C-terminal domain (FIG. 2a).Whilst the A and B pocket domains were poorly acetylated, the C-terminaldomain exhibited much stronger acetylation (FIG. 2b), suggesting thatthis region contains the major sites for acetylation. By studying theacetylation of an extensive panel of mutant derivatives, the majorregions of acetylation were mapped to between residue 794 to 829, and830 to 884 (FIG. 2c). A C-terminal derivative from residue 881 and 928,which contains seven lysine residues, failed to be acetylated (FIGS. 2eand f). Thus, the sites of acetylation occur on lysine residues withinthe 87 residue region flanked by residue 794 to 880. Altogether, eightlysines occur within this region, and later in this report we identifytwo lysine residues from this region that are acetylated.

[0080] D. The E1A Oncoprotein Enhances Acetylation.

[0081] The adenovirus E1A oncoprotein binds to and sequesters a varietyof cellular proteins, including pRb and p300 family members, throughdomains that are necessary for E1A to exert its effects on cellulargrowth and differentiation 3. The binding of p300/CBP proteins requiresan N-terminal domain in E1A, and two domains, referred to as conservedregions (CR) 1 and 2, for binding to pRb3. Given the well-documentedinteraction of p300 and pRb with E1A, together with the p300-dependentacetylation of pRb described here (FIG. 1), it was of interest toinvestigate the influence of E1A on the acetylation level of pRb. Inorder to assess the effect of E1A, recombinant wild-type E1A wastitrated into the pRb acetylation reaction, together with p3001135-2414as the source of HAT, which also harbours the E1A-binding domain forp30011. Upon the addition of E1A protein there was a significant andspecific stimulation in pRb acetylation mediated by the p300 HAT (FIG.3a). This effect was concentration-dependent since at high molar ratiosof E1A to p300 and pRb, further increases in E1A levels caused the lossof enhancement of pRb acetylation (FIG. 3ci). Since E1A can bind to bothpRb and p300 proteins3, these results are consistent with the idea thatE1A stimulates the acetylation of pRb by recruiting pRb and p300 into aternary complex containing E1A, pRb and p300, thus directing the p300HAT activity to pRb. The loss of enhancement of pRb acetylation at highlevels of E1A may therefore result from E1A forming complexesindependently with pRb or p300.

[0082] In contrast to its effect upon pRb, the presence of E1A failed tosignificantly affect p300-autoacetylation, and the p300-dependentacetylation of core histones was marginally affected by E1A (two-fold atmost; FIG. 3b). However, the acetylation of the p53 tumour suppressorprotein and E2F-1, the activities of which are known to be influenced byacetylation 12, 13, 14, 15, was compromised in the presence of E1A(FIGS. 3aii and cii). Overall, these results establish that E1A canalter the p300-dependent acetylation of target protein substrates and,most importantly, augment the level of pRb acetylation.

[0083] E. Physical Interaction of E1A with pRb is Required.

[0084] To determine the specificity of the effects on pRb, we assessedthe properties of a variety of mutant derivatives in E1A that arecompromised in ability to bind either pRb or p300 (FIG. 4a). Mutant E1Aproteins that failed to bind pRb, namely ΔCR1, ΔCR2 and pm928, or p300,such as ΔN3616, could not effectively stimulate the p300-dependentacetylation of pRb, whilst there was little effect on theautoacetylation of p300 (FIGS. 4a, b, and c; and data not shown),arguing that a physical interaction between all three proteins isnecessary for E1A to stimulate pRb acetylation by the p300 HAT activity.

[0085] Moreover, since pRb is acetylated in 293 cells (FIG. 1e), whichexpress E1A17, it was of interest to compare the level of pRbacetylation in 293 cells with other cell-types that do not express E1A.By immunoprecipitation, we found that pRb in 293 cells showed a muchhigher level of acetylation compared to other cell-types, includingU2OS, T98G and C33A (FIG. 4d; and data not shown). Since 293 cellsexpress the E1A protein, the results support the idea that E1Astimulates the acetylation of pRb by p300. A further consistency withthis idea was the presence of p300 in the pRb immunoprecipitate, whichwas also seen in the 293 cell immunocomplex (FIG. 4d).

[0086] F. pRb Acetylation Enhances Interaction with MDM2.

[0087] To elucidate the functional importance of pRb acetylation, wewere prompted to assess its impact on the MDM2 oncoprotein, which bindsto the C-terminal region of pRb and as a result antagonises thepRb-dependent down-regulation of E2F activity 7. As expected fromprevious studies 7, in a biochemical assay pRb bound to MDM2 and theminimal region in pRb responsible for the interaction was mapped towithin residue 792 to 928 (data not shown). To determine the importanceof pRb acetylation in the regulation of MDM2 binding, pRb was acetylatedby the p300 HAT and thereafter acetylated pRb purified as described.Then, the binding efficiency of in vitro translated MDM2 was assessed.At equivalent levels of input pRb, we found that the efficiency ofbinding between MDM2 and acetylated pRb was much greater than to thenon-acetylated pRb (FIG. 4e), suggesting that MDM2 preferentially bindsto the acetylated form of pRb. Similar experiments performed to evaluatethe influence of acetylation on the interaction of pRb with E2F failedto detect any differences.

[0088] If the acetylation of pRb facilitates its interaction with MDM2,we would predict that the pRb/MDM2 complex should be detectable in acell-type in which a high level of pRb acetylation was apparent. To testthis idea, we compared the levels of the pRb/MDM2 complex in 293 cells,in which pRb is acetylated, to other cells where pRb acetylation couldnot be detected (FIG. 4d). Significant levels of human MDM2 weredetectable in pRb immunoprecipitates performed from 293 cells, butundetectable in the other cell-types under study (FIG. 4f). This resultis consistent with the conclusion that acetylated pRb binds efficientlyto MDM2, and the presence of acetylated pRb in 293 cells.

[0089] Conversely, conditions that enhance acetylation would be expectedto promote the interaction of pRb with MDM2. To this end, we treatedT98G cells with trichostatin A (TSA), which blocks histone deacetylase(HDAC) activity 18 and thereafter assessed the interaction between pRband MDM2. Whereas the pRb/MDM2 complex was not apparent in T98G cells,it was clearly detectable in extracts prepared from T98G cells treatedwith TSA (FIG. 4g). Overall, these results are consistent with the ideathat acetylation influences the interaction between pRb and MDM2.

[0090] G. Lys 873 and 874 of pRb are Targets for Acetylation.

[0091] To further clarify the role of acetylation in pRb, we identifiedlysine residues that are directly acetylated by the p300 HAT andthereafter assessed their functional importance for wild-type pRb. Wefocussed our attention on the C-terminal region between residue 830 and884, which contains five lysine residues and is acetylated by p300 HAT(FIG. 2e). By systematically altering individual lysine to arginineresidues by site-directed mutagenesis, we identified lysines 873 and 874within a lysine rich region as subject to acetylation (FIG. 5a). Thus,acetylation of pRb from residue 830 to 884 was lost upon alteringresidues 873 and 874 to arginine (FIG. 5a). In turn, this observationsuggests that the remaining three lysine residues within this region ofpRb are unlikely to be acetylated by p300 HAT.

[0092] H. Acetylation of pRb Reduces Phosphorylation by cdks.

[0093] As we were interested to determine the functional importance ofacetylation in pRb we considered that acetylation may influence pRbphosphorylation, which provides a major level of control in regulatingpRb activity [1, 2]. Furthermore, the C-terminal region between residue830 to 884 has been suggested to contain a motif that influences pRbphosphorylation by cyclin-dependent kinases (cdk) [19]. To test thisidea, we introduced 873/874RR into wild-type pRb and thereafter assessedphosphorylation of the 873/874RR mutant by cyclin E/cdk2, which actsduring the G1 phase to phosphorylate pRb [1, 3]. In addition, weprepared 873/874QQ in which lysine residues 873/874 were altered toglutamine residues (FIG. 5b). In contrast to 873/874RR, where thearginine residue retains the basic charge of the lysine residue butcannot be acetylated, a change to a glutamine residue would beanticipated to mimic the acetylation status of the lysine residue [20].In SAOS2 cells both wild-type pRb and 873/874RR were phosphorylated toan equivalent level by exogenous cyclin E/cdk2, whereas 873/874QQ failedto reach a comparable level of phosphorylation (FIG. 5biii). A similaranalysis was performed in U2OS cells which contain high levels ofendogenous cdk activity. As for SAOS2 cells, both wild-type pRb and873/874RR were phosphorylated to an equivalent level, whereas 873/874QQwas significantly reduced (FIG. 5biv). These results suggest that theacetylation of pRb on lysine residue 873 and 874 influences thephosphorylation control of pRb activity.

[0094] I. Acetylation Provides a New Level of pRb Control.

[0095] The acetylation of pRb described here suggests a new level ofcontrol in the regulation of pRb activity, and a novel mechanism ofaction through which viral oncoproteins can overcome tumour suppressoractivity. Whilst the acetylation of nucleosomes is recognised to play animportant role in regulating chromatin accessibility [5, 6, 18], andsome DNA binding transcription factors are known to be acetylated [13,14, 21, 22, 23, 24], to our knowledge the acetylation control of pRbprovides the first example of an acetylation-regulated and cellcycle-relevant protein-protein interaction. Our findings point towards amechanism whereby E1A can direct the p300 HAT activity to enzymaticallymodify pRb and thereafter alter pRb function (FIG. 6), thus suggestingthat E1A can act as a targeting subunit for a cellular HAT activitywhich thereafter enforces altered growth-control.

[0096] Since the p300-binding domain in E1A is necessary for some of thephysiological effects ascribed to E1A, such as the induction of DNAsynthesis and inhibition of differentiation [3], and p300 may functionin proliferation control in normal cells [5], it is a possibility thatthe ability of E1A to stimulate pRb acetylation is responsible for someof the effects previously ascribed to the p300-binding domain [3, 25].

[0097] In this respect, our results imply that pRb acetylationinfluences the interaction with the MDM2 oncoprotein, which maysubsequently allow MDM2 to exert some of its oncogenic growth-promotingeffects through the release of E2F activity [7]. Thus, the ability ofE1A to stimulate pRb acetylation and thereafter MDM2 binding to pRbwould be expected to translate into a pathway that augments cell cycleprogression.

[0098] J. Acetylation of pRb may Counterbalance HDAC Activity.

[0099] It is noteworthy that HDAC binds to pRb, and may contribute topRb-dependent growth control [26]. An interesting possibility is thatthe acetylation of pRb may to a certain extent be counterbalanced byHDAC, thus maintaining pRb in a hypo-acetylated state. It is consistentwith this idea that we also found that HDAC can efficiently deacetylatepRb, and further that TSA treatment which blocks HDAC activity augmentsthe interaction between pRb and MDM2 (FIG. 4).

[0100] K. Conclusions.

[0101] In addition to the above, it is of considerable interest that theacetylation of pRb may influence the ability of phosphorylation by cdkkinases to modulate pRb tumour suppressor function. We note that lysineresidues 873 and 874 are part of a previously identified cdk dockingsite [19] which, if the properties of such a site were to be modulatedby acetylation, provides a plausible mechanism for the influence ofacetylation on pRb phosphorylation. The data presented here support theidea that the acetylation of pRb impedes subsequent phosphorylation bycdk kinases, implying perhaps that the acetylation control of pRb may bea regulatory pathway that acts in a fundamentally distinct fashion tothat of pRb phosphorylation.

[0102] In conclusion, our results suggest a rationale for thesequestration of p300 by E1A, namely in directing a cellular enzyme tothe control of tumour suppressor activity (FIG. 6). These observationstherefore establish a novel relationship between p300, pRb andacetylation in which E1A acts as an enzyme targeting subunit in anenzyme-substrate-type of relationship, thus favouring the enzymaticmodification of pRb in a fashion that contributes to a loss ofpRb-dependent growth control.

[0103] Material and Methods.

[0104] Plasmids.

[0105] pGEX-Rb, Δ21 and Δ22 mutants, pGEX-Rb 763-928, pGEX-Rb 794-829,844, 857, 864, 876, 884, 896, 910 were as previously described [9, 19].pGEX-Rb 830-884, 830-928, 881-928, and 641-775 were cloned by direct PCR(see primers below) from pcDNA3-9E10-Rb, and pGEX-Rb 379-656 containedthe Nhe1 to EcoR1 fragment from pGEX-Rb9. pGEX E1A, pm928 and Δ36 werecloned by PCR (see primers below) from pCMV-E1A, pCMV-E1Apm928 orpCMV-E1AΔ2-36 [16, 27]. The His-p3001195-1673 and Flag-p3001135-2414bacterial expression vector and Flag-p300 (wild-type) were as described[28, 29]. pcDNA-MDM2 has been described [30]. Plasmids 5′-primer3′-primer GST-Rb 5′-cgggatccagaatcttagtatcaattgg-3′5′-cggaattctcattcatctgatccttcaatatc-3′ (830-884) (SEQ ID NO:1) (SEQ IDNO:2) GST-Rb 5′-cgggatccagaatcttagtatcaattgg-3′5′-cggaattctcatttctcttccttgtttg-3′ (830-928) (SEQ ID NO:3) (SEQ ID NO:4)GST-Rb 5′-cgggatccggatcagatgaagcagatg-3′5′-cggaattctcatttctcttccttgtttg-3′ (881-928) (SEQ ID NO:5) (SEQ ID NO:6)GST-Rb 5′-ccattgaaatctacctctc-3′ 5′-tcacctggtggaagcatacctgc-3′ (641-775)(SEQ ID NO:7) (SEQ ID NO:8) GST-E1A 5′-cgggatccatgagacatattatctgccac-3′5′-ccctcgagttatggcctggggcgtttac-3′ (SEQ ID NO:9) (SEQ ID NO:10) GST-5′-cgggatcccattttgaaccacctacc-3′ 5′-ccctcgagttatggcctggggcgtttac-3′ E1A(ΔN36) (SEQ ID NO:11) (SEQ ID NO:12)

[0106] In Vitro Protein Acetylation Assay.

[0107] PAGE analysis of protein acetylation was performed as described[29], with slight modification. The indicated amounts of input proteinswere incubated at 30 C for 45-60 min in 30 μl of volume with 5×HAT assaybuffer (250 mM Tris pH 8.0, 25% glycerol, 0.5 mM EDTA, 250 mM KCl, and10 mM sodium butyrate), 90 pmol [14C]acetyl CoA (55 mCi/mol, AmershamLife Science Inc.) and the appropriate amount of water to a final volumeof 30 μl. After incubation, reactions were stopped by adding 15 μl of3×SDS loading buffer (150 mM Tris-HCl (pH 6.8), 6% SDS, 30% glycerol,0.3% bromophenol blue, 3% mercaptoethanol), and analysed on SDS-PAGE.The gels were dried and exposed for autoradiography for 48-96 h.Quantitation of acetylation was performed by phosphoimaging.

[0108] Expression and Purification of Glutathione-S-transferase FusionProteins.

[0109] Glutathione-S-transferase fusion protein expression andpurification was performed as recommended by the manufacturer(Pharmacia). Fresh overnight cultures of BL21(DE3) pLys (Invitrogen)transformed with the appropriate pGEX-recombinants were diluted 1:10 inLuria-Bertani (LB) medium containing ampicillin (100 μg/μg) andincubated at 37° C. with shaking. After 2 h of growth,isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma) was added to finalconcentration of 0.5 mM The cultures were subsequently incubated at 30 Cfor 4-5 h for protein expression before harvesting.

[0110] For fusion protein purification using glutathione-Sepharose(Pharmacia), bacterial cultures were pelleted by centrifugation at 6,000rpm for 10 min at 4 C. The pellets were resuspended in cold PBS (Sigma,5 ml PBS/100 ml of bacterial culture). The bacteria were then lysed onice by mild sonication and Triton-X-100 (Sigma) was added to the lysateto a final concentration of 1%. The lysate was incubated at 4 C for 30min, then centrifuged at 13,000 rpm, 4 C for 30 min. The bacterialsupernatants were rocked for 30-45 min at 4 C with glutathione-Sepharose(200 μl of beads/100 ml culture). The glutathione-Sepharose beads werewashed three times with 20 ml of cold PBS supplemented with 1% ofTriton, and once with 20 ml of cold PBS. For the analysis of boundproteins, the appropriate amount of beads were boiled in 1× samplebuffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromophenolblue, 1% mercaptoethanol), and loaded onto an SDS-polyacrylamide gel.Proteins were visualized by Coomassie blue staining. To elute theGST-fusion proteins from beads, proteins were eluted in elution buffer(50 mM Tris-HCl (pH 8.0), 10 mM reduced glutathione, 120 mM NaCl). Allfusion proteins were subsequently dialysed into BC100 buffer (20 mMTris-HCl (pH 8.0), 0.5 mM EDTA, 100 mM KCl, 20% glycerol, 0.5 mM DTT,0.5 mM PMSF).

[0111] Expression and Purification of His-Tagged Fusion Proteins.

[0112] The procedure for His-tagged fusion protein purification wassimilar to the GST-fusion protein purification. Briefly, 5 ml of PBS(supplemented with 0.5M NaCl, 10 mM imidazole pH 7.4 and proteaseinhibitor cocktail (Calibiochem)) was used to resuspend the bacterialpellet from 100 ml culture. The bacteria were then lysed on ice by mildsonication and pelleted at 13,000 rpm for 30 min at 4 C. The supernatantwas collected and incubated with Ni-NBT agarose (QIAGEN) at 4 C for 1 h.After incubation, the beads were washed 5 times in PBS (supplementedwith 0.5M NaCl, 40 mM imidazole pH 7.4). Elution was carried out usingBC100 buffer supplemented with 200 mM imidazole.

[0113] Production of Flag-p3001135-2414 Baculovirus.

[0114] To generate the baculovirus vector for Flag-p3001135-2414, theBAC-To-BAC Baculovirus Expression system from Gibco Life Technologieswas used. In short, Flag-p3001134-2414 sequence was taken out from thebacterial expression vector [34] by RsrII and NotI digest. The fragmentisolated was cloned directly into the RsrII and NotI sites ofpFastBacHTa vector to generate the baculovirus vector expressingFlag-p3001135-2414.

[0115] Expression and Purification of Flag-p300 from sf9 cells. Toexpress flag-p300 in sf9 cells, 1.5×107 sf9 cells were infected with theappropriate baculovirus at multiplicity of infection of 10. The sf9cells were harvested for protein purification after 48 h of incubationat 25 C. The method of purification was essentially as described [31].The sf9 cells were pelleted by mild centrifugation at 700 rpm and washedtwice with cold PBS. The sf9 cells were lysed in 2 ml of HEMG buffer (25mM Hepes-KOH (pH 7.6), 0.1 mM EDTA, 12.5 mM MgCl2, 10% glycerol)supplemented with 400 mM KCl, 0.1% NP-40 and protease inhibitor cocktail(Calibiochem). The samples were quickly freeze-thawed three times andcentrifuged at 13,000 rpm at 4 C for 30 min. The supernatant wascollected and incubated with anti-flag M2 affinity gel (Sigma, 200 μl ofbeads/ml of supernatant), in HEMG buffer containing 200 mM KCl. After 2h of incubation at 4 C, the beads were washed 5 times with HEMG buffersupplemented with 200 mM KCl. Proteins bound on the beads were elute inelution buffer which is basically HEMG buffer supplemented with 160 mMKCl and 1 mg/ml of flag-peptide (Sigma). Elution was carried out at 4 Cfor 2 h. Proteins elute were analysed by SDS-PAGE and visualized byCoomassie blue staining.

[0116] Tissue Culture and Transfection.

[0117] The C33A, U2OS, SAOS2, T98G, HEK293, and A31 cells were allcultured in Dulbecco modified Eagle medium (Gibco) supplemented with 10%foetal bovine serum at 37 C in 5% CO₂. Transfections were carried outusing calcium phosphate method as described [32].

[0118] Western Blot and Immunoprecipitation.

[0119] The following antisera were used; anti-Ac-Lys (New EnglandBiolabs), anti-Ac-H4 (Serotec), anti-Rb monoclonal IF8, G3-245(Pharmingen) and C15 (Santa Cruz), anti-p300 N15 (Santa Cruz) andanti-MDM2H221 (Santa Cruz).

[0120] For immunoprecipitation of endogenous acetylated pRb, cellpellets collected from SAOS2, U2OS, T98G, A31, and HEK293 cells werelysed in IPH buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% NP-40,protease inhibitor cocktail (Roche), 5mM EDTA, 5 μM TSA (Sigma)). Theprotein concentration of the extracts was determined by Bradford assay(BioRad). Protein-A-agarose was first incubated with anti-Rb antibodyIF8 and C15 for 1-2 h at 4 C. The beads were washed 3 times with IPHbuffer, and incubated with cell extracts overnight at 4 C. Proteinsbound to the beads were elute with 3×SDS loading buffer and analysed byWestern blotting using appropriated antibodies. To immunoprecipitate pRbfrom transfected cells, SAOS2 cells were transfected with pcDNA3-Rb orempty vector as described 32 and, after 48 h, harvested andimmunoprecipitated with IF8 and C15.

[0121] In vitro Binding Assay Between Rb and MDM2.

[0122] About 2 μg of GST-Rb was acetylated by Flag-p300 in a standard 30μl reaction as described above, but using 1 μl of 10 mM cold acetyl-CoA.For the unacetylated Rb sample, the acetylation reaction was carried outwithout acetyl-CoA. After completion of the acetylation reactions, thereaction was incubated with Flag beads (Sigma) in 400 μl of IPH bufferto remove Flag-p300. The supernatant was collected andimmunoprecipitated with protein-A-agarose as described above with eitheranti-Ac-Lys antibody or anti-Rb antibody (C15 and IF8) to purify theacetylated Rb and unacetylated Rb respectively; the anti-acetylatedlysine antibody failed to recognise the unacetylated pRb (FIG. 1c). Invitro transcribed and translated MDM2 was incubated with equal amounts(determined by immunoblotting) of acetylated or unacetylated Rb toaccess binding efficiency. After incubation, the beads were washed threetimes with IPH buffer (containing 0.25% NP-40), and samples analysed onSDS-PAGE followed by Western blot analysis with the MDM2 antibody H221.

[0123] Site-Directed Mutagenesis and Transfection.

[0124] Site-directed mutagenesis was performed using Quickchange(Strategene). PCR was performed using pcDNA3-9E10 Rb to create pcDNA3-Rb(873/874RR) and pcDNA-Rb (873/874QQ), and pGEX-Rb 830-884 to generatepGEX-Rb 830-884/873/874RR. All constructs were confirmed by sequencing.Transfection into SAOA2 and U2OS cells in the presence or absence ofcyclin E/cdk2 kinase and immunoblotting was carried out as previouslydescribed [32].

DETAILED DESCRIPTION OF THE FIGURES

[0125]FIG. 1

[0126] The retinoblastoma Protein is Modified by Acetylation.

[0127] a) Schematic representation of GST-pRb, pRbΔ21 and pRbΔ22 used inthe acetylation assays.

[0128] b) i) The indicated GST-pRb derivatives were expressed, purifiedand analysed on an SDS gel. The gel was stained with coomassie blue.

[0129] ii) The indicated GST proteins (2 μg) were incubated withhis-tagged p3001195-1673 (about 0.5 μg) and 14C acetyl CoA as describedand analysed by SDS PAGE.

[0130] c) GST-pRb (2 μg) was incubated with Flag-tagged p3001135-2414(0.3 μg) in the presence or absence of acetyl CoA as indicated. Afterincubation, 1/10 of the reaction was loaded onto tracks 1 and 3, and9/10 of the reaction onto 2 and 4. The upper panel shows an immunoblotperformed with the anti-pRb C15 polyclonal antibody and the lower panelan immunoblot performed with anti-acetyl lysine antisera.

[0131] d) SAOS2 (Rb−/−) cells were transfected with pCMV-Rb (30 μg;tracks 2, 4, 5 and 6; indicated by +) or the empty vector pCMV (30 μg;tracks 1 and 3; indicated by −) as described. At 48 h, cell extractswere prepared and immunoprecipitated with anti-pRb monoclonal antibody(tracks 1, 2, 3 and 4) or a control monoclonal antibody (tracks 5 and6). Samples were resolved by SDS PAGE and immunoblotted with eitheranti-pRb monoclonal antibodies (tracks 1, 2 and 6) or the anti-acetyllysine antisera (tracks 3, 4 and 5); pRb is indicated.

[0132] e) Extracts prepared from 293 cells were immunoprecipitated withanti-pRb (C15 and IF-8; tracks 2 and 5) or control antibodies (tracks 3and 6), and after electrophoresis, immunoblotted with either anti-pRbmonoclonal antibody G3-245 (tracks 2 and 3) or anti-acetyl lysineantisera (tracks 5 and 6). Tracks 1 and 4 show that input 293 extractimmunoblotted with either anti-pRb or anti-acetyl lysine.

[0133]FIG. 2

[0134] The C-Terminal Region of pRb is a Major Site of Acetylation.

[0135] The indicated derivatives of pRb (a, c and e) were expressed andpurified as GST-fusion proteins and analysed on an SDS gel (bi and di).Each GST-pRb derivative (2 to 3 μg) was assessed for in vitroacetylation by Flag-tagged p3001135-2414 (0.3 μg) in the presence of 14Cacetyl CoA as described (bii, dii and f). Acetylated pRb andauto-acetylated p300 is indicated (bii, dii and f), and GST alone inputis shown (bi; track 1 with the GST protein indicated by •. Note that theRb(A) fragment shows a low level of acetylation (bii, track 3).

[0136]FIG. 3

[0137] Adenovirus E1A Augments the p300-Dependent Acetylation of the pRbProtein.

[0138] a) In vitro acetylation assay in which the indicated recombinantproteins (coomassie blue stained gel shown in i)) were assessed forlevel of acetylation (shown in ii) in the presence of 14C acetyl CoA. Anequal amount of Flag-tagged p3001135-2414 (0.3 μg) was used throughout,together with GST-pRb (2 μg; tracks 1, 2 and 3) or GST-E2F-1 (2 μg;tracks 4, 5 and 6) and increasing His-E1A13S (0.3 or 1 μg in tracks 2and 5, and 3 and 6 respectively). The same gel is shown, representingprotein levels in i) and 14C acetylation in ii).

[0139] b) In vitro 14C acetylation assay in which Flag-taggedp3001135-2414 (0.3 μg) together with increasing His-E1A13S (0.3 or 1 μgin tracks 2 and 3 respectively) was incubated with purified chicken corehistones (1 μg; tracks 1, 2 and 3). The same gel is shown, representingprotein levels in i) and 14C acetylation in ii).

[0140] c) In vitro 14C acetylation assay to compare the effect of E1A onpRb (i) and p53 (ii) acetylation, in which Flag-tagged p3001135-2414(0.3 μg) was incubated with either wild-type GST-tagged pRb (1.0 μg; i))or GST-p53 (0.5 μg; ii) in the presence of increasing levels ofGST-E1A12S (0.04, 0.2, 1 or 5 μg in tracks 2 to 5). In track 6, theeffect of GST (5 μg) is shown. Note that the assays shown in i) and ii)were performed in parallel in the same experiment.

[0141]FIG. 4

[0142] Domains in E1A for p300-Dependent Acetylation of pRb andAcetylated pRb Binds to MDM2.

[0143] a) In vitro acetylation assay in which Flag-tagged p3001135-2414(0.3 μg) together with GST-pRb (2 μg) was incubated with eitherwild-type GST-E1A, GST-Δ36 or GST-pm928 (1 μg) as indicated. The effectof the buffer (track 5) or GST (1 μg; track 1) alone was also assessed.The quantification of the level of p300 autoacetylation (b) and pRbacetylation (c) in the presence of the E1A derivatives is shown. Thehighest level of acetylation was given an arbitrary value of 100. E1Aderivatives GST-ΔCR1 and GST-ΔCR2 failed to stimulate pRb acetylation,and • indicates a polypeptide that is likely to be a derivative of p300.

[0144] b) Extracts prepared from the indicated cells (SAOS2, A31, U2OS,T98G and 293) were immunoprecipitated with anti-pRb (C15 and IF-8)antibodies and, after electrophoresis, immunoblotted with eitheranti-pRb G3-245 (top), anti-p300 N15 (middle) or anti-acetylated lysine(bottom) antisera. Note that although equivalent levels of pRb areimmunoprecipitated from the different cell extracts, only 293 cells(which express E1A), contain high levels of p300 (middle) and acetylatedpRb (bottom) in the immunocomplex; C15 and IF8 do not immunoprecipitatemurine pRb in A31 cells.

[0145] c) GST-pRb was acetylated with Flag-p300 (wild-type) andthereafter Flag-p300 removed. The binding of in vitro translated MDM2 tonon-acetylated (track 2) or acetylated pRb (track 3) was assessed asdescribed followed by immunoblotting with anti-MDM2. The input MDM2 isshown in track 1, and tracks 4 and 5 show the input (5%) GST-pRb with(track 5) or without (track 4) acetylation determined by immunoblottingwith anti-pRb (G3-245). Note that the binding of MDM2 to acetylated pRbwas much more efficient than the binding activity for non-acetylatedpRb.

[0146] d) Extracts prepared from the indicated cells (SAOS2, U2OS, T98Gand 293) were immunoprecipitated with anti-pRb (C15 and IF-8) antibodiesand, after electrophoresis, immunoblotted with either anti-pRb G3-245(top) or anti-MDM2 (bottom) monoclonal antibody. Note that a high levelof co-immunoprecipitated MDM2 is seen in the pRb immunocomplex only from293 cell extracts, and that U2OS and T98G cells show very low butdetectable levels (after longer exposure) of co-immunoprecipitated MDM2.

[0147] e) Extracts prepared from T98G cells either treated with orwithout TSA (5 μM) for 18-20 h, and untreated SAOS2 cells wereimmunoprecipitated with anti-pRb (C15 and IF8) antibodies and afterelectrophoresis immunoblotted with either anti-pRb G3-245 (top) oranti-MDM2 (bottom) monoclonal antibody. Note the presence of MDM2 in thepRb immunocomplex from TSA-treated T98G cells.

[0148]FIG. 5

[0149] Functional Effects of pRb Acetylation.

[0150] a) In vitro acetylation of the indicated GST derivatives (2 μg)of pRb, namely pRb763-928, pRb830-884 and 873/874RR (i) after expressionand purification as GST proteins (ii) and 14C acetylation (iii) withFlag-tagged p3001135-2414 (0.3 μg).

[0151] b) In vivo phosphorylation of the indicated derivatives offull-length pRb, namely wild-type pRb, 873/874RR and 873/874QQ (ii)after transfection into SAOS2 (iii) or U2OS (iv) cells of pcDNA3-9E10Rb,pcDNA3-Rb (873/874RR) or pcDNA3-Rb (873/874QQ) (10 μg) in the presenceof exogenous cyclin E/cdk2 (4 μg of each vector [33]; indicated by +).After 24 h of transfection, extracts were prepared, quantitated, andimmunoblotted with anti-pRb (G3-245). The position of hyper (+) and hypo(−) phosphorylated (P) pRb is indicated. i) shows the sequencesurrounding lysine 873/874 as previously noted 19.

[0152]FIG. 6

[0153] Acetylation Control of pRb Activity.

[0154] It is envisaged that the E1A oncoprotein recruits p300 and pRbinto a ternary complex to facilitate the p300-dependent acetylation ofpRb. The results suggest that the acetylation of pRb favours theinteraction with MDM2, which thereafter releases E2F from pRb control.Subsequently, E2F is able to promote cell cycle progression.

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[0187] Although the foregoing invention has been described in somedetail by way of illustration and example for the purposes of clarityand understanding, it will be readily apparent to those of skill in theart that certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

1. An assay for a modulator of acetylation of pRb by p300, whichcomprises: a) bringing into contact a p300 protein, a pRb protein and aputative modulator compound under conditions where the p300 protein, inthe absence of said modulator is capable of acetylating the pRb protein;and b) providing conditions for acetylation of said pRb protein; c)measuring the degree of inhibition of acetylation caused by saidmodulator compound.
 2. An assay according to claim 1 wherein said pRb isin the form of a fusion protein with a detectable tag.
 3. An assayaccording to claim 1 or 2 wherein the adenovirus E1A protein is present.4. An assay according to any one of the preceding claim wherein the pRbis in the form of a C-terminal fragment of at least 40 amino acidscomprising at least one target lysine residue.
 5. An assay according toany one of the preceding claims wherein said p300 protein is a fragmentof at least about 450 amino acids of a full-length p300, said fragmentretaining the ability to acetylate pRb.
 6. An assay according to any oneof the preceding claims which is a cell based assay in which one or bothof said p300 and pRb proteins are expressed using a recombinant DNAconstruct.
 7. An assay according to any one of the preceding claimswhich further includes the step of phosphorylating the pRb protein usingcyclinE/cdk2 as a means to determine the acetylation of pRb.
 8. Amodulator obtained by an assay of any one of the preceding claims.
 9. Anassay for a modulator of cell cycle progression which comprises: a)bringing into contact an acetylated pRb protein and an MDM2 proteinunder conditions where, in the absence of modulator, the two proteinsbind to each other; b) providing a putative modulator; and c)determining the degree of modulation of binding of the two proteinscaused by said modulator.
 10. An assay for a modulator of acetylation ofa p53 protein which assay comprises: a) providing a p300 protein and anE1A protein under conditions in which the acetylation activity of thep300 protein can be redirected to a pRb protein; and b) providing a p53protein and a putative modulator compound under conditions in which saidp300 protein, in the absence of said modulator and said E1A protein, canacetylate p53; and c) determining the effect of the presence of said E1Aand said modulator on acetylation of p53.
 11. A compound obtained by theassay method of claim 10.